Fatty acids are important components of lipids such as phospholipids, triacylglycerols, etc. Fatty acids containing two or more unsaturated bonds are collectively referred to as polyunsaturated fatty acids (PUFA) are known to specifically include arachidonic acid, dihomo-γ-linolenic acid, eicosapentaenoic acid, docosahexaenoic acid, etc. Some of these polyunsaturated fatty acids cannot be synthesized in the animal body. Thus, it is necessary to take such polyunsaturated fatty acids as essential amino acids through food.
In the animal body, polyunsaturated fatty acids are distributed in a wide variety of organs and tissues. For example, arachidonic acid has been separated from lipids extracted from the animal adrenal gland or liver. However, polyunsaturated fatty acids are contained in small quantities in the animal organs, and the extraction and separation of polyunsaturated fatty acids from the animal organs are not sufficient to supply a large quantity of polyunsaturated fatty acids. For this reason, methods of acquiring polyunsaturated fatty acids by culturing various microorganisms have been developed. Among those microorganisms, a Mortierella microorganism is known as a microorganism capable of producing lipids containing polyunsaturated fatty acids such as arachidonic acid, etc. Furthermore, an attempt to produce polyunsaturated fatty acids in plants has also been made. Polyunsaturated fatty acids constitute storage lipids such as triacylglycerols, etc. and are known to be accumulated within the cells of microorganisms or in the seeds of plants.
Triacylglycerols, which are storage lipids, are produced in vivo as follows. Acyl transfer occurs on glycerol-3-phosphate by glycerol-3-phosphate acyltransferase to form lysophosphatidic acid. Next, acyl transfer further occurs by lysophosphatidic acid acyltransferase to form phosphatidic acid. This phosphatidic acid is, in turn, dephosphorylated by phosphatidic acid phosphatase to form diacylglycerol. Finally, acyl transfer occurs by diacylglycerol acyltransferase to form triacylglycerol.
In the triacylglycerol biosynthesis pathway or the phospholipid biosynthesis pathway described above, it is known that the acylation reaction of glycerol-3-phosphate to form lysophosphatidic acid is mediated by glycerol-3-phosphate acyltransferase (hereinafter sometimes referred to as “GPAT”; EC 2.3.1.15).
The presence of GPAT genes has been reported so far in several organisms. As mammalian GPAT genes, two types of microsome (membrane-bound) and mitochondria (membrane-bound) have been cloned (Non-Patent Literature 2). Likewise, three types of microsome (membrane-bound), mitochondria (membrane-bound) and chloroplast (free) have also been cloned as plant GPAT genes (Non-Patent Literature 3).
As the GPAT genes derived from the fungus Saccharomyces cerevisiae, two types of microsomal (membrane-bound) GPT2/GAT1 (YKR067w) and SCT1/GAT2 (YBL011w) have been cloned; it is known that simultaneous deletion of both genes results in lethality (Non-Patent Literature 4). In these fungal genes, it is shown that GPT2 has the activity to use a wide range of fatty acids from palmitic acid (16:0) to oleic acid (18:1) as substrate, whereas SCT1 has a strong selectivity in using 16 carbon fatty acids such as palmitic acid (16:0) and palmitoleic acid (16:1)) as substrate (Non-Patent Literature 4).
Furthermore, the GPAT genes have also been cloned from many other organism species. Above all, the GPAT derived from the microorganisms of the genus Mortierella capable of producing lipids is reported as follows.
In the GPAT derived from Mortierella ramanniana, microsomal GPAT has been isolated and shown to be used as an acyl donor with a 5.4-fold higher selectivity of oleic acid (18:1) than palmitic acid (16:0) (Non-Patent Literature 5). It is reported that the GPAT derived from Mortierella alpina (hereinafter sometimes referred to as “M. alpina” has a glycerol-3-phosphate acyltransferase activity in its microsomal fraction (Non-Patent Literature 6).
It is shown that when the GPAT present in the microsome of M. alpina (in a membrane-bound state) is reacted in vitro with various acyl CoAs, the GPAT uses as substrate a broad range of polyunsaturated fatty acids including oleic acid (18:1), linoleic acid (18:2), dihomo-γ-linolenic acid (DGLA) (20:3) and arachidonic acid (20:4) (Patent Literature 1).
It is shown that when the GPAT cloned from M. alpina (ATCC #16266) (hereinafter referred to as MaGPAT1 (ATCC#16266)) was expressed in transformant Yarrowia lipolytica designed to enable biosynthesis to give eicosapentaenoic acid (EPA), in total fatty acids, the composition of dihomo-γ-linolenic acid (DGLA) (20:3) increased and the composition of oleic acid (18:1) decreased. The results indicate that polyunsaturated fatty acid with a longer chain length and high degree of unsaturation is selectively incorporated (Patent Literature 2).
In recent years, it is reported that GPAT homologue or MaGPAT2 was isolated from M. alpina (1S-4) and showed the substrate specificity different from MaGPAT1 (Patent Literature 3). That is, it is suggested that MaGPAT1 would show high specificity to palmitic acid and MaGPAT2 would show high specificity to oleic acid.